In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

The structurally related T cell surface molecules CD28 and CTLA-4 interact with cell surface ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APC) and modulate T cell antigen recognition. Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (K d values ∼12 and ∼200 nM, respectively) that are high when compared with other molecular interactions that contribute to T cell–APC recognition. In the present study, we use surface plasmon resonance to measure the affinity and kinetics of CD80 binding to CD28 and CTLA-4. At 37°C, soluble recombinant CD80 bound to CTLA-4 and CD28 with K d values of 0.42 and 4 μM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (koff); sCD80 dissociated from CD28 and CTLA-4 with koff values of ⩾1.6 and ⩾0.43 s−1, respectively. Such rapid binding kinetics have also been reported for the T cell adhesion molecule CD2 and may be necessary to accommodate dynamic T cell–APC contacts and to facilitate scanning of APC for antigen.

Most asymptomatic individuals infected with HIV-1 have a cytotoxic T lymphocyte (CTL) response to the virus Gag proteins which can be demonstrated in vitro. Epitopes have been mapped in p17 Gag and p24 Gag restricted by HLA-B8 (p17-3 and p24-13) and -B27 (p24-14). Viruses isolated from patients who make CTL responses to these peptides vary within the genetic sequences encoding these epitopes and some mutations lead to reduction in killing activity in vitro. This was attributed to either failure of the variant epitope to bind major histocompatibility complex class I or failure of T-cell receptors to bind the presented peptide. But peptide variants of class I-restricted epitopes cause 'antagonism', that is, the presence of a variant epitope (in the form of peptide) inhibits normal lysis of targets presenting the original epitope. This mirrors similar findings in class II-restricted systems. Here we report that naturally occurring variant forms of p17-3, p24-13 and p24-14 may cause antagonism of CTL lines derived from the same individuals. The effect is present if the epitopes are derived from synthetic peptides and when they are processed from full-length proteins expressed by either recombinant vaccinia constructs or replicating HIV.